Spontaneous Dilated Cardiomyopathy Research Articles

GFPT1 deficiency is a novel cause of a dilated cardiomyopathy and is required for myocardial adaptation to chronic pressure-overload stress-induced heart failure

Abstract Background GFPT1 is the rate-limiting enzyme of a pathway called the hexosamine biosynthesis pathway (HBP) that turns fructose 6-phosphate derived from glucose into another monosaccharide called N-acetylglucosamine (GlcNAc). GlcNAc is an important substrate for modifying diverse cellular proteins on their serine and threonine residues termed O-GlcNAcylation often competing with phosphorylation and altering their activity or stability (Bond & Hanover; 2015). Increased GFPT1 activity is known to stimulate the cellular integrated stress response improving protein quality control and cell survival (Denzel et al 2014). In the heart, GFPT1 expression and O-GlcNAcylation increases in the early stages of chronic cardiac stress but the physiological role of GFPT1 in regulating heart function including its role during chronic stress remains unclear. Identifying molecular pathways that promote adaptive cardiac remodelling is a promising approach to discover novel therapies for heart failure. Methods Cardiomyocyte-specific GFPT1 knockout mice were generated to study the effects of GFPT1 deletion in hearts. Heart function was examined by echocardiography. HBP activity was quantified using Western blotting of O-GlcNAc-modified proteins. Abdominal aortic banding (AAB) was used to induce chronic pressure-overload heart failure. Results Homozygous GFPT1 KO mice spontaneously develop a dilated cardiomyopathy (DCM) with a 30% mortality rate (ejection fraction 32% compared with 62% for floxed controls and end diastolic volume 125 microL versus 60 microL; n=5-6 per group; p <0.05). Hearts showed reduced levels of total protein O-GlcNAcylation suggesting impaired hexosamine biosynthesis pathway activity. Heart function could be rescued by supplementing their diets with oral glucosamine bypassing GFPT1 to provide metabolites required for hexosamine biosynthesis and normalising O-GlcNAcylation (ejection fraction improved to 40% in KO with supplementation versus KO without; n=4-6 per group; p <0.05). Heterozygous KO mice do not develop a DCM but when subject to pressure-overload cardiac stress, develop worse heart failure with a 50% mortality rate over 9 weeks of pressure-overload (ejection fraction 43% in het KO versus 53% in floxed control; n= 3-6 per group; p = 0.0025). Conclusion Deficiency of GFPT1 is a novel cause of a spontaneous dilated cardiomyopathy. GFPT1 is required for physiological growth of the heart that may in part involve the O-GlcNAcylation of proteins. Partial GFPT1 deficiency is associated with adverse cardiac remodelling following pressure-overload stress. Increasing GFPT1 activity may be a key therapeutic target in developing novel heart failure treatments.

Jmjd4 Facilitates Pkm2 Degradation in Cardiomyocytes and Is Protective Against Dilated Cardiomyopathy.

A large portion of idiopathic and familial dilated cardiomyopathy (DCM) cases have no obvious causal genetic variant. Although altered response to metabolic stress has been implicated, the molecular mechanisms underlying the pathogenesis of DCM remain elusive. The JMJD family proteins, initially identified as histone deacetylases, have been shown to be involved in many cardiovascular diseases. Despite their increasingly diverse functions, whether JMJD family members play a role in DCM remains unclear. We examined Jmjd4 expression in patients with DCM, and conditionally deleted and overexpressed Jmjd4 in cardiomyocytes in vivo to investigate its role in DCM. RNA sequencing, metabolites profiling, and mass spectrometry were used to dissect the molecular mechanism of Jmjd4-regulating cardiac metabolism and hypertrophy. We found that expression of Jmjd4 is significantly decreased in hearts of patients with DCM. Induced cardiomyocyte-specific deletion of Jmjd4 led to spontaneous DCM with severely impaired mitochondrial respiration. Pkm2, the less active pyruvate kinase compared with Pkm1, which is normally absent in healthy adult cardiomyocytes but elevated in cardiomyopathy, was found to be drastically accumulated in hearts with Jmjd4 deleted. Jmjd4 was found mechanistically to interact with Hsp70 to mediate degradation of Pkm2 through chaperone-mediated autophagy, which is dependent on hydroxylation of K66 of Pkm2 by Jmjd4. By enhancing the enzymatic activity of the abundant but less active Pkm2, TEPP-46, a Pkm2 agonist, showed a significant therapeutic effect on DCM induced by Jmjd4 deficiency, and heart failure induced by pressure overload, as well. Our results identified a novel role of Jmjd4 in maintaining metabolic homeostasis in adult cardiomyocytes by degrading Pkm2 and suggest that Jmjd4 and Pkm2 may be therapeutically targeted to treat DCM, and other cardiac diseases with metabolic dysfunction, as well.

GLP-1 analog liraglutide-induced cardiac dysfunction due to energetic starvation in heart failure with non-diabetic dilated cardiomyopathy

BackgroundGlucagon-like peptide-1 (GLP-1) reduces cardiovascular events in diabetic patients; however, its counter-protective effects have also been suggested in patients with heart failure and the clear explanation for its mechanisms have not yet been offered.MethodsThe effects of GLP-1 analog on cardiac function and energy metabolism, especially glycemic and lipid metabolisms were elucidated using non-diabetic J2N-k hamsters which showed spontaneous dilated cardiomyopathy. J2N-k hamsters were treated with PBS (HF group), low-dose (HF-L group) or high-dose liraglutide (HF-H group).ResultsIn failing heart, GLP-1 analog exerted further deteriorated cardiac function (e.g. positive and negative dP/dt; p = 0.01 and p = 0.002, respectively) with overt fibrosis and cardiac enlargement (heart/body weight, 5.7 ± 0.2 in HF group versus 7.6 ± 0.2 in HF-H group; p = 0.02). The protein expression of cardiac muscles indicated the energy starvation status. Indirect calorimetry showed that failing hearts consumed higher energy and carbohydrate than normal hearts; moreover, this tendency was augmented by GLP-1 analog administration. Upon 10% glucose solution loading with GLP-1 analog administration (HF-H-G group) as complementary experiments, the cardiac function and fibrosis significantly ameliorated, whereas carbohydrate utilization augmented further and lipid utilization reduced more. The prognosis of HF-H-G group also significantly improved (p = 0.025).ConclusionsGlucagon-like peptide-1 analog caused the relative but desperate shortage of glycemic energy source for the failing cardiac muscles and it may restrict ATP synthesis, resulting in cardiac function deterioration. Therefore, appropriate energy supply and amount of carbohydrate intake should be carefully considered when administrating incretin-related drugs to patients with heart failure.

Cartilage Oligomeric Matrix Protein: Matricellular and Matricrine Signaling in Cardiovascular Homeostasis and Disease.

Cardiovascular (CV) diseases remain a leading cause of morbidity and mortality in the world. Increasing the understanding of the pathogenesis of various CV diseases may provide novel therapeutic targets to improve their prevention and treatment. Cartilage oligomeric matrix protein (COMP), also known as thrombospondin-5 (TSP-5), is a matricellular protein that is abundantly expressed in both cartilage and the CV system. Our group and others have identified COMP as playing critical roles in maintaining CV homeostasis. COMP, expressed and produced by vascular smooth muscle cells (VSMCs), maintains VSMC contractile phenotypes. COMP deficiency enhances VSMC migration and aggravates VSMC calcification and atherosclerosis. Moreover, a lack of COMP leads to spontaneous dilated cardiomyopathy in mice. COMP is also secreted by platelets in circulating blood and negatively regulates haemostasis and thrombosis. A series of COMP binding proteins, such as integrin α7β1, integrin β3, thrombin, and bone morphogenetic protein 2, have been identified in the CV system, and they have been determined to mediate various COMP functions. The matrix metalloproteinase (A Disintegrin and Metalloproteinase with Thrombospondin motifs) ADAMTS-7 is a regulatory enzyme that is responsible for the degradation of COMP in the CV system. ADAMTS-7 expression correlates with atherosclerosis and vascular calcification in both human genome-wide association studies and in vivo mice models via COMP-dependent and COMP-independent mechanisms. In this review, we summarize what is currently known about the matricellular and matricrine signaling of COMP mediated by its respective binding partners as well as its proteolytic regulation by ADAMTS-7 in CV disease.

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy.

Cardiosphere‐derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non‐ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c‐kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.

Mechanistic Role of Thioredoxin 2 in Heart Failure.

Thioredoxin 2 (Trx2) is a pivotal mitochondrial protein that regulates redox signaling. The mitochondrial Trx2 is expressed ubiquitously, but it is found at the highest levels in metabolically active tissues like the heart. Global gene knockout of Trx2 results in embryonic lethality, likely due to the increased cellular oxidative stress. Moreover, mice with cardiac-specific Trx2 deletion develop spontaneous dilated cardiomyopathy (DCM), correlating with increased apoptosis stress kinase-1 (ASK1) signaling and increased cardiomyocyte apoptosis. Cardiomyocyte apoptosis is a common mechanism in the pathogenesis of heart failure. Our results show that Trx2 is essential for maintaining cardiac function. In this chapter, we summarize the key mechanistic role of Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species (ROS) generation and by inhibiting ASK1-dependent apoptosis in heart failure. Trx2 and ASK1 represent promising targets to develop therapeutic strategies for the treatment of DCM and heart failure.

The E3 ubiquitin ligase c-Cbl mediates integrin β1 ubiquitination during dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is characterized by dilatation of the ventricular chambers and impaired myocardial contractility. The results of our previous study indicated that a deficiency in matricellular cartilage oligomeric matrix protein (COMP) led to spontaneous and progressive DCM in mice via the ubiquitination/degradation of integrin β1. However, the specific ubiquitin enzyme involved in degradation of integrin β1 and the pathogenesis of DCM remain elusive. We first compared gene expression profiles in hearts from 3-month-old wild type and COMP−/− mice using microarray analysis. Among the E3 ubiquitin ligases upregulated in COMP−/− hearts, c-Cbl silencing rescued the ubiquitination/degradation of integrin β1, myofilament loss, apoptosis and connexin-43 deficiency in cardiomyocytes due to the silencing of COMP. Furthermore, c-Cbl silencing by intramyocardial injections of siRNA into 1-month-old COMP−/− mice ameliorated spontaneous DCM in vivo, as evidenced by the inhibition of the dilation of ventricular chambers, impaired ejection fraction and myofilament loss. A subsequent cellular ubiquitination assay revealed that overexpression of c-Cbl induced ubiquitination of integrin β1, whereas the G306E mutation in c-Cbl, which prevented the binding of c-Cbl to its substrates, had no effect on integrin β1 ubiquitination, indicating that c-Cbl directly caused the ubiquitination of integrin β1 in the hearts. In conclusion, our results demonstrate that c-Cbl mediates the ubiquitination/degradation of integrin β1, which leads to COMP deficiency-induced DCM.

Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function.

Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation. Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

2014 AHA Late-Breaking Basic Science Abstracts

### 24178 ### MAVS Mediates Protection against Myocardial Ischemic Injury with Hydrogen Sulfide David Durrant, Adolfo G Mauro, Juan Valle Raleigh, Anindita Das, Jun He, Khoa Nguyen, Stefano Toldo, Antonio Abbate, Fadi N Salloum; Virginia Commonwealth Univ, Richmond, VA Background : Hydrogen sulfide (H2S) has been shown to protect against myocardial ischemic and inflammatory injury in part by preserving mitochondrial integrity. Since mitochondrial antiviral signaling (MAVS) protein has been implicated in attenuating Bax-mediated cytochrome c release from mitochondria caused by oxidative stress or ischemia, we sought to determine whether MAVS mediates the cardioprotective effects of H2S. Methods and Results : After baseline echocardiography, adult male wild type (WT) or MAVS KO mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min. followed by 24 h reperfusion. Mice were pretreated with Na2S (100 μg/kg; ip ) or saline 1 h before MI. Infarct size, measured with TTC staining, was reduced and LV fractional shortening (FS) was preserved with Na2S at 24h post MI in WT mice as compared to saline-treated mice, but not in MAVS KO mice (Figs. A and B). The risk area was not different between the groups. Western blot analysis revealed a significant decline in myocardial MAVS expression at 24h post MI, which was preserved with Na2S (Fig. C). Another subset of mice was subjected to permanent coronary artery occlusion and treated with Na2S or saline daily for 28 days. LVFS decreased significantly at 28 days post-MI in the saline group, but was significantly preserved with Na2S (Fig. D). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na2S group (22.4 ± 2.7%) as compared to control (33.5 ± 2.1%, P<0.05). Survival rate was 2 fold higher with Na2S compared to saline (P<0.05). Western blot analysis confirmed a significant …

Melusin gene therapy: a novel approach to fight familial dilated cardiomyopathy

We have previously demonstrated that melusin, a muscle specific chaperone protein, is required for cardiac compensatory response to stress conditions (Brancaccio et al 2003) and its overexpression effectively counteracts cardiac dilation and heart failure induced by aortic stenosis (De Acetis et al 2005). Chaperones are cytoprotective proteins that recognize a broad panel of proteins partially damaged by stress conditions or genetic mutations allowing them to retain correct function. Using and in vivo gene therapy approach with cardiotropic adeno associated virus 9 vector we show that overexpression of melusin effectively counteract dilated cardiomyopathy in a mouse model mimicking human Emery Dreyfus familial cardiomyopathy caused by LaminA H222P mutation. These mice develop spontaneous dilated cardiomyopathy with age and males die within 8-10 month of age due conduction defect and sudden death. One month old homozygous mutant mice were injected with AAV9-Melusin or with AAV9-LacZ as control (I.V. injection of 1012 viral particles/mouse) and their cardiac function was monitored by echocardiography for the following 8 months. While mice injected with control virus progressively developed left ventricle dilation and reduced contractility (FS%), mice injected with AAV9-Melusin retained physiological level of contractility and were protected toward LV dilation. More interestingly, 8 month after the treatment, 40% of control mutant mice died as described by the literature, while Melusin-treated mice were 100% alive. Biochemical analysis indicated that melusin binds LaminA in cardiomyocyte, suggesting that chaperone function is crucial for the protective effect. These data indicate that melusin gene therapy has a strong potential to treat familial cardiomyopathy.

Abstract 210: Thioredoxin-2 Inhibits Mitochondrial Ros Generation And Ask1 Activity To Maintain Cardiac Function

Background: Increasing evidence indicates that mitochondrial-derived reactive oxygen species (ROS) and cellular apoptosis contribute to the pathogenesis of cardiac dysfunction. Mitochondrial thioredoxin (Trx2) is a key protein regulating cellular redox and survival, However, but its role in normal cardiac growth has not been determined. Methods and Results: We have generated cardiac-specific Trx2 knockout mice (Trx2-cKO) to determine the physiological importance of the Trx2 system in the heart. Trx2-cKO mice developed a spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, fibrosis, reduced ventricular wall thickness, and progressive contractile dysfunction, resulting in death due to heart failure by 4 months of age. Cardiac changes in Trx2-cKO mice were accompanied by disruption of mitochondrial integrity and function, as evident by alterations in mitochondrial number, ultrastructure, membrane potential and ATP production. Increases in ASK1 signaling and ROS production preceded mitochondrial damage, cellular apoptosis and contractile dysfunction in both Trx2-cKO hearts and isolated cardiomyocytes. Moreover, deletion of ASK1 attenuates ROS production, mitochondrial dysfunction and cellular apoptosis in Trx2-deficient cardiomyocytes. These data indicate that ASK1 is a major target of Trx2 and that activation of ASK1 is causally associated with mitochondrial dysfunction, ROS production and cellular apoptosis. We also detected reduced Trx2 expression and increased ASK1 activity in human hearts from patients with cardiomyopathy, suggesting that this mechanism is clinically important. Conclusion: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial ROS production and ASK1-dependent apoptosis. These results suggest that pharmacological inhibition of ASK1 may provide a therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

Discovering novel targets for autoantibodies in dilated cardiomyopathy

There is increasing evidence that a large proportion of dilated cardiomyopathy (DCM) cases are mediated by autoimmune processes. Since DCM is a fatal disorder with rapid aggravation and is the leading cause of heart transplantation, further insights into disease pathogenesis are needed. Recent studies have separated the pathogenic capacity of autoantibodies and initial clinical trials removing such autoantibodies via immunoadsorption have been promising. In order to elucidate the full autoantibody repertoire involved in DCM, we applied an autoantibody screening test using ventricular and atrial proteomes as autoantigenic sources and subsequently tested the autoantibody-binding patterns of sera from dogs with spontaneous DCM. With this method, we detected five potentially DCM-related autoantigens which were identified by MS as being: myosin heavy chain cardiac muscle alpha isoform, alpha cardiac actin, mitochondrial aconitate hydratase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and brain glycogen phosphorylase (GPBB). The recovery of two known DCM autoantigens (myosin heavy chain and alpha cardiac actin) and the discovery of three novel autoantigens (mitochondrial aconitate hydratase, GADPH, and GPBB) underscore the efficacy of this experimental method and the significance of the spontaneous canine DCM model.

Assessment of Regional Systolic and Diastolic Myocardial Function Using Tissue Doppler and Strain Imaging in Dogs with Dilated Cardiomyopathy

Background: Tissue Doppler Imaging (TDI) or strain (St) imaging could provide sensitive indices for early detection and treatment follow‐up of canine dilated cardiomyopathy (DCM). Analysis of TDI and St features in dogs with overt DCM is a prerequisite before using these new criteria in prospective screenings of predisposed families or in clinical trials. Hypothesis: Radial and longitudinal right and left myocardial motion, assessed by TDI and St variables, is altered in dogs with DCM. Animals: Case records for 26 dogs; 14 with DCM and 12 healthy controls of comparable age and weight were reviewed. Methods: A retrospective analysis was conducted of conventional echocardiography, 2‐dimensional color TDI, and St imaging data. Results: The DCM group was characterized by decreases in radial and longitudinal systolic velocity gradients of the left ventricular free wall (LVFW), radial and longitudinal absolute values of peak systolic St of the LVFW, and longitudinal systolic right ventricular (RV) velocities (all P &lt; .001 versus control) associated with longitudinal postsystolic contraction waves in 7/14 dogs. Early diastolic LVFW velocities also were decreased for longitudinal (P &lt; .01) and radial (P &lt; .05) motions. All radial LVFW, longitudinal basal LVFW, and RV systolic velocities were negatively correlated with heart rate (P &lt; .01). Conclusions and Clinical Importance: LV contractility along both the short and long axes is impaired in dogs with spontaneous DCM, as is systolic RV and diastolic LVFW function. These myocardial alterations are associated with an inverse force‐frequency relationship. Studies now are needed to determine the comparative sensitivity of TDI and St variables for the early detection of canine DCM.

Assessment of Regional Systolic and Diastolic Myocardial Function Using Tissue Doppler and Strain Imaging in Dogs with Dilated Cardiomyopathy

Tissue Doppler Imaging (TDI) or strain (St) imaging could provide sensitive indices for early detection and treatment follow-up of canine dilated cardiomyopathy (DCM). Analysis of TDI and St features in dogs with overt DCM is a prerequisite before using these new criteria in prospective screenings of predisposed families or in clinical trials. Radial and longitudinal right and left myocardial motion, assessed by TDI and St variables, is altered in dogs with DCM. Case records for 26 dogs; 14 with DCM and 12 healthy controls of comparable age and weight were reviewed. A retrospective analysis was conducted of conventional echocardiography, 2-dimensional color TDI, and St imaging data. The DCM group was characterized by decreases in radial and longitudinal systolic velocity gradients of the left ventricular free wall (LVFW), radial and longitudinal absolute values of peak systolic St of the LVFW, and longitudinal systolic right ventricular (RV) velocities (all P < .001 versus control) associated with longitudinal postsystolic contraction waves in 7/14 dogs. Early diastolic LVFW velocities also were decreased for longitudinal (P < .01) and radial (P < .05) motions. All radial LVFW, longitudinal basal LVFW, and RV systolic velocities were negatively correlated with heart rate (P < .01). LV contractility along both the short and long axes is impaired in dogs with spontaneous DCM, as is systolic RV and diastolic LVFW function. These myocardial alterations are associated with an inverse force-frequency relationship. Studies now are needed to determine the comparative sensitivity of TDI and St variables for the early detection of canine DCM.

Distinct functions of junD in cardiac hypertrophy and heart failure

Cardiac hypertrophic stimuli induce both adaptive and maladaptive growth response pathways in heart. Here we show that mice lacking junD develop less adaptive hypertrophy in heart after mechanical pressure overload, while cardiomyocyte-specific expression of junD in mice results in spontaneous ventricular dilation and decreased contractility. In contrast, fra-1 conditional knock-out mice have a normal hypertrophic response, whereas hearts from fra-1 transgenic mice decompensate prematurely. Moreover, fra-1 transgenic mice simultaneously lacking junD reveal a spontaneous dilated cardiomyopathy associated with increased cardiomyocyte apoptosis and a primary mitochondrial defect. These data suggest that junD promotes both adaptive-protective and maladaptive hypertrophy in heart, depending on its expression levels.

Calcium-activated force in a turkey model of spontaneous dilated cardiomyopathy: Adaptive changes in thin myofilament Ca 2+ regulation with resultant implications on contractile performance

Using saponin skinned fibers, we investigated whether decreased myofilament calcium responsiveness and contractile activation may in part contribute to heart failure in an animal model of idiopathic spontaneous cardiomyopathy (SCM). We addressed the question as to whether there are adaptive changes at the level of the thin myofilaments in turkey poults with SCM. The calcium concentration ([Ca 2+]) required for 50% activation ([Ca 2+] 50%) was 0.80±0.12 μ m ( n=12) vs. 0.76±0.08 μ m ( n=12) and the Hill coefficient was 1.98±0.20 ( n=12) vs. 2.14±0.38 ( n=12) for control and SCM muscles respectively. Maximal Ca 2+-activated force was not different between control fibers and fibers from failing hearts (3.83±0.88 g/mm 2 vs. 3.65±0.39 g/mm 2). These data indicate there are no differences in calcium-activation between fibers from control and failing myocardium. The effects of caffeine, an agent that increases myofilament Ca 2+ sensitivity, were also studied. Addition of 10 m m caffeine resulted in a 0.06 pCa unit leftward shift of the force-pCa relationship in control hearts and 0.14 pCa units in SCM hearts. Caffeine (30 m m) increased force by 26±2.1% ( n=7) in control fibers and 44.5±8.7% ( n=8) in myopathic fibers at a pCa of 6.0. The increased responsiveness of muscles from failing hearts to caffeine indicates adaptive changes at the level of the thin myofilaments. Addition of dibutyryl-3′,5′-cyclic-Adenosine Monophosphate (D-cAMP) resulted in a 0.21 pCa rightward shift on the calcium axis to higher intracelluar calcium concentrations in control myocardium and 0.38 pCa units in SCM failing myocardium. The muscles were also sinusoidally oscillated at frequencies ranging between 0.01 and 100 Hz. In this analysis the frequency at which dynamic stiffness is minimum is taken as a measure of cross-bridge cycling rate. In control muscles, the frequency of minimum stiffness (f min) was 1.20±0.11 ( n=4) whereas it was 0.71±0.08 Hz ( n=4) in myopathic muscles. The addition of 10 μ m D-cAMP shifted f min from 1.20±0.11 Hz to 1.68±0.09 Hz (Δ= 0.48±0.06) in control fibers whereas in SCM fibers it caused greater shift of f min from 0.71±0.08 Hz to 1.73±0.08 Hz (Δ= 1.02±0.07). This differential effect of D-cAMP indicates adaptive changes at the level of the myofilaments. Our studies indicate that adaptive changes at the level of the thin myofilaments can potentially alter the “apparent” calcium-force relationship in failing myocardium and contractile performance, and that failing myocardium has the potential to generate similar levels of force as normal non-failing myocardium.

THE USE OF M‐MODE ECHOCARDIOGRAPHY IN DETERMINING CARDIAC OUTPUT IN DOGS WITH NORMAL, LOW, AND HIGH OUTPUT STATES: COMPARISON TO THERMODILUTION METHOD

The utility of M‐mode echocardiographic determination of left ventricular volume and cardiac output was evaluated by comparing four commonly used formulae for ventricular volume, derived from end‐systolic (ESD) and end‐diastolic (EDD) ventricular dimensions. Cardiac index (CI) was calculated by the following formulae, where HR = heart rate and BSA = body surface area (M2): 1. (EDD3‐ ESD3) * HR/BSA; 2. (0.85 * EDD3 ‐ 1.2 *ESD3) * HR/BSA; 3. (EDD3.16 ‐ ESD3.16) * HR/BSA; and 4. {[7/(2.4 + EDD)] * [EDD3] ‐ [7/(2.4 + ESD)] * [ESD3)}* HR/BSA. Cardiac index, obtained invasively by thermodilution, was compared to values determined echocardiographically in normal dogs (Group 1) and in three states of altered cardiac function: after positive inotropic stimulation with graded doses (0,5,10, and 15 μg/kg/min) of intravenous dobutamine (Group 2); with experimentally induced heartworm disease (Group 3); and with spontaneous dilated cardiomyopathy (Group 4). Cardiac index, determined echocardiographically, was plotted against CI, determined with thermodilution, and regression equations calculated. In Group 1 (n = 15), a significant relationship (P &lt; 0.01) was observed with each of the four formulae (r2= 0.54‐0.68), however, individual estimates of CI were often inaccurate. With inotropic stimulation (Group 2;3 normal dogs, n = 12 data points), the relationship was significant (P &lt; 0.05), but the coefficients of determination were less than in the unstimulated normal dogs (r2= 0.41‐0.47). No significant relationship was seen between CI, determined echocardiographically and CI, determined by thermodilution, when overt cardiac disease was present (Groups 3 and 4; n = 7 and 13, r2= 0.13‐0.21, respectively). Specific formulae were evaluated for accuracy by comparing their coefficients of determination and their ability to predict mean and individual measured CI. Using the paired Student's t‐test, CI determined noninvasively were shown to differ significantly (P &lt; 0.05) between methods and between formulae in many instances. No one formula was found to be consistently more accurate than the others. We conclude that, in normal dogs, while echocardiographic estimation of CI is significantly related to CI measured with thermodilution, individual variation diminishes clinical utility. However, this noninvasive technique should prove useful in comparing populations with minimal or no cardiac dysfunction. Secondly, the relationship is diminished, but not lost, after inotropic stimulation. M‐mode echocardiographic estimation of CI offers little promise for accurate assessment of cardiac performance in cardiac disease states. Lastly, although none of the four formulae for CI compared herein were found to be consistently superior to the other three, formulae 3 and 4 appear to be most useful in estimating CI in normal dogs.