Contribution of skeletal muscle-specific microRNA-133b to insulin resistance in heart failure.

Insulin resistance (IR) is one of the hallmarks of heart failure (HF). Abnormalities in skeletal muscle (SM) metabolism have been identified in patients with HF. However, the underlying mechanisms of IR development in SM in HF are poorly understood. Herein, we hypothesize that HF upregulates miR-133b in SM and in turn alters glucose metabolism and the propensity toward IR. Mitochondria isolated from SM of mice with HF induced by transverse aortic constriction (TAC) showed lower respiration and downregulation of muscle-specific components of the tricarboxylic acid (TCA) cycle, AMP deaminase 1 (AMPD1), and fumarate compared with those from control animals. RNA-Seq and subsequent qPCR validation confirmed upregulation of SM-specific microRNA (miRNA), miR-133b, in TAC versus sham animals. miR-133b overexpression alone resulted in significantly lower mitochondrial respiration, cellular glucose uptake, and glycolysis along with lower ATP production and cellular energy reserve compared with the scramble (Scr) in C2C12 cells. miR-133b binds to the 3'-untranslated region (UTR) of KLF15, the transcription factor for the insulin-sensitive glucose transporter, GLUT4. Overexpression of miR-133b lowers GLUT4 and lowers pAkt in presence of insulin in C2C12 cells. Finally, lowering miR-133b in primary skeletal myocytes isolated from TAC mice using antagomir-133b reversed the changes in KLF15, GLUT4, and AMPD1 compared with the scramble-transfected myocytes. Taken together, these data demonstrate a role for SM miR-133b in altered glucose metabolism in HF and suggest the therapeutic potential in HF to improve glucose uptake and glycolysis by restoring GLUT4 abundance. The data uncover a novel mechanism for IR and ultimately SM metabolic abnormalities in patients with HF.NEW & NOTEWORTHY Heart failure is associated with systemic insulin resistance and abnormalities in glucose metabolism but the underlying mechanisms are poorly understood. In the skeletal muscle, the major peripheral site of glucose utilization, we observe an increase in miR-133b in heart failure mice, which reduces the insulin-sensitive glucose transporter (GLUT4), glucose uptake, and metabolism in C2C12 and in myocytes. The antagomir for miR-133b restores GLUT4 protein and markers of metabolism in skeletal myocytes from heart failure mice demonstrating that miR-133b is an exciting target for systemic insulin resistance in heart failure and an important player in the cross talk between the heart and the periphery in the heart failure syndrome.

Mitochondrial Creatine Kinase Attenuates Pathologic Remodeling in Heart Failure.

BACKGROUND: Abnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF. METHODS: First, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice. RESULTS: In people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling. CONCLUSIONS: In the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

Skeletal muscle ATP kinetics are impaired in frail mice.

The interleukin-10 knockout mouse (IL10(tm/tm)) has been proposed as a model for human frailty, a geriatric syndrome characterized by skeletal muscle (SM) weakness, because it develops an age-related decline in SM strength compared to control (C57BL/6J) mice. Compromised energy metabolism and energy deprivation appear to play a central role in muscle weakness in metabolic myopathies and muscular dystrophies. Nonetheless, it is not known whether SM energy metabolism is altered in frailty. A combination of in vivo (31)P nuclear magnetic resonance experiments and biochemical assays was used to measure high-energy phosphate concentrations, the rate of ATP synthesis via creatine kinase (CK), the primary energy reserve reaction in SM, as well as the unidirectional rates of ATP synthesis from inorganic phosphate (Pi) in hind limb SM of 92-week-old control (n = 7) and IL10(tm/tm) (n = 6) mice. SM Phosphocreatine (20.2 ± 2.3 vs. 16.8 ± 2.3 µmol/g, control vs. IL10(tm/tm), p < 0.05), ATP flux via CK (5.0 ± 0.9 vs. 3.1 ± 1.1 µmol/g/s, p < 0.01), ATP synthesis from inorganic phosphate (Pi → ATP) (0.58 ± 0.3 vs. 0.26 ± 0.2 µmol/g/s, p < 0.05) and the free energy released from ATP hydrolysis (∆G ∼ATP) were significantly lower and [Pi] (2.8 ± 1.0 vs. 5.3 ± 2.0 µmol/g, control vs. IL10(tm/tm), p < 0.05) markedly higher in IL10(tm/tm) than in control mice. These observations demonstrate that, despite normal in vitro metabolic enzyme activities, in vivo SM ATP kinetics, high-energy phosphate levels and energy release from ATP hydrolysis are reduced and inorganic phosphate is elevated in a murine model of frailty. These observations do not prove, but are consistent with the premise, that energetic abnormalities may contribute metabolically to SM weakness in this geriatric syndrome.

Creatine kinase-overexpression improves myocardial energetics, contractile dysfunction and survival in murine doxorubicin cardiotoxicity.

Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5 mg/kg) administration using in vivo (31)P MRS and (1)H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity.

Creatine kinase overexpression improves ATP kinetics and contractile function in postischemic myocardium.

Reduced myofibrillar ATP availability during prolonged myocardial ischemia may limit post-ischemic mechanical function. Because creatine kinase (CK) is the prime energy reserve reaction of the heart and because it has been difficult to augment ATP synthesis during and after ischemia, we used mice that overexpress the myofibrillar isoform of creatine kinase (CKM) in cardiac-specific, conditional fashion to test the hypothesis that CKM overexpression increases ATP delivery in ischemic-reperfused hearts and improves functional recovery. Isolated, retrograde-perfused hearts from control and CKM mice were subjected to 25 min of global, no-flow ischemia and 40 min of reperfusion while cardiac function [rate pressure product (RPP)] was monitored. A combination of (31)P-nuclear magnetic resonance experiments at 11.7T and biochemical assays was used to measure the myocardial rate of ATP synthesis via CK (CK flux) and intracellular pH (pH(i)). Baseline CK flux was severalfold higher in CKM hearts (8.1 ± 1.0 vs. 32.9 ± 3.8, mM/s, control vs. CKM; P < 0.001) with no differences in phosphocreatine concentration [PCr] and RPP. End-ischemic pH(i) was higher in CKM hearts than in control hearts (6.04 ± 0.12 vs. 6.37 ± 0.04, control vs. CKM; P < 0.05) with no differences in [PCr] and [ATP] between the two groups. Post-ischemic PCr (66.2 ± 1.3 vs. 99.1 ± 8.0, %preischemic levels; P < 0.01), CK flux (3.2 ± 0.4 vs. 14.0 ± 1.2 mM/s; P < 0.001) and functional recovery (13.7 ± 3.4 vs. 64.9 ± 13.2%preischemic RPP; P < 0.01) were significantly higher and lactate dehydrogenase release was lower in CKM than in control hearts. Thus augmenting cardiac CKM expression attenuates ischemic acidosis, reduces injury, and improves not only high-energy phosphate content and the rate of CK ATP synthesis in postischemic myocardium but also recovery of contractile function.

Creatine kinase-mediated improvement of function in failing mouse hearts provides causal evidence the failing heart is energy starved.

ATP is required for normal cardiac contractile function, and it has long been hypothesized that reduced energy delivery contributes to the contractile dysfunction of heart failure (HF). Despite experimental and clinical HF data showing reduced metabolism through cardiac creatine kinase (CK), the major myocardial energy reserve and temporal ATP buffer, a causal relationship between reduced ATP-CK metabolism and contractile dysfunction in HF has never been demonstrated. Here, we generated mice conditionally overexpressing the myofibrillar isoform of CK (CK-M) to test the hypothesis that augmenting impaired CK-related energy metabolism improves contractile function in HF. CK-M overexpression significantly increased ATP flux through CK ex vivo and in vivo but did not alter contractile function in normal mice. It also led to significantly increased contractile function at baseline and during adrenergic stimulation and increased survival after thoracic aortic constriction (TAC) surgery-induced HF. Withdrawal of CK-M overexpression after TAC resulted in a significant decline in contractile function as compared with animals in which CK-M overexpression was maintained. These observations provide direct evidence that the failing heart is "energy starved" as it relates to CK. In addition, these data identify CK as a promising therapeutic target for preventing and treating HF and possibly diseases involving energy-dependent dysfunction in other organs with temporally varying energy demands.

Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery.

OBJECTIVES: The aim of this study was to quantify acute myocardial retention of cardiac-derived stem cells (CDCs) and evaluate different delivery methods with positron emission tomography (PET). BACKGROUND: Success of stem cell transplantation for cardiac regeneration is partially limited by low retention/engraftment of the delivered cells. A clinically applicable method for accurate quantification of cell retention would enable optimization of cell delivery. METHODS: The CDCs were derived from syngeneic, male Wistar Kyoto (WK) rats labeled with [(18)F]-fluoro-deoxy-glucose ((18)FDG) and injected intramyocardially into the ischemic region of female WK rats after permanent left coronary artery ligation. The effects of fibrin glue (FG), bradycardia (adenosine), and cardiac arrest were examined. Imaging with (18)FDG PET was performed for quantification of cell retention. Quantitative polymerase chain reaction (PCR) for the male-specific SRY gene was performed to validate the PET results. RESULTS: Myocardial retention of cells suspended in phosphate-buffered saline 1 h after delivery was 17.6 +/- 11.5% by PCR and 17.8 +/- 7.3% by PET. When CDCs were injected immediately after induction of cardiac arrest, retention was increased to 75.6 +/- 18.6%. Adenosine slowed the ventricular rate and doubled CDC retention (35.4 +/- 5.3%). A similar increase in CDC retention was observed after epicardial application of FG at the injection site (37.5 +/- 8.2%). The PCR revealed a significant increase in 3-week cell engraftment in the FG animals (22.1 +/- 18.6% and 5.3 +/- 3.1%, for FG and phosphate-buffered saline, respectively). CONCLUSIONS: In vivo PET permits accurate measurement of CDC retention early after intramyocardial delivery. Sealing injection sites with FG or lowering ventricular rate by adenosine might be clinically translatable methods for improving stem cell engraftment in a beating heart.

Preservation of cardiac contractility after long-term therapy with oxypurinol in post-ischemic heart failure in mice.

Previously, we showed that oral allopurinol increased survival in mice with post-ischemic cardiomyopathy and attributed this outcome to an improvement of excitation-contraction coupling that boosted contractility. In this study, we tested the sustainability of this enhanced contraction associated with decreased oxidative damage over an extended time. Mice were divided into three groups: sham-operated control, myocardial infarction-heart failure (MI-HF), and oxypurinol-treated heart failure (Oxy-HF). After 9-11 months, echocardiography showed that mice treated with oxypurinol (1mM in drinking water) had significantly higher left ventricle fractional contraction and fractional wall thickening during systole than did mice in the MI-HF group (left ventricle fractional contraction: 28.4+/-2.2% vs. 19.9+/-2.3%, P<0.05; left ventricle fractional wall thickening: 45.0+/-4.0% vs. 23.5+/-2.0%, P<0.05). Left ventricular diastolic dimension, however, remained enlarged (0.50+/-0.04 vs. 0.54+/-0.05 cm, not significant). Twitch force was significantly higher at any given external Ca(2+) concentration in the Oxy-HF group than in the MI-HF group (P<0.01); amplitudes of intracellular Ca(2+) transients were also higher in the Oxy-HF group but were not statistically different from those of the MI-HF group. Force-frequency relation was improved in the Oxy-HF group. Muscle in the Oxy-HF group exhibited increases in myofilament Ca(2+) responsiveness, as evidenced by significantly higher maximal Ca(2+)-activated force (77.8+/-12.7 vs. 36.4+/-4.4 mN/mm(2), P<0.01). Finally, lipid peroxidation and myofilament oxidation were suppressed in the Oxy-HF group. These results indicate that the beneficial effects of antioxidation can be sustained by long-term treatment with oxypurinol after ischemic heart failure, with significantly improved cardiac contractility.

Ectopic expression of the sodium-iodide symporter enables imaging of transplanted cardiac stem cells in vivo by single-photon emission computed tomography or positron emission tomography.

OBJECTIVES: We examined the sodium-iodide symporter (NIS), which promotes in vivo cellular uptake of technetium 99m ((99m)Tc) or iodine 124 ((124)I), as a reporter gene for cell tracking by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. BACKGROUND: Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo. METHODS: The human NIS complementary deoxyribonucleic acid was transduced into rat cardiac-derived stem cells (rCDCs) using lentiviral vectors. Rats were injected intramyocardially with up to 4 million NIS(+)-rCDCs immediately after left anterior descending coronary artery ligation. Dual isotope SPECT (or PET) imaging was performed, using (99m)Tc (or (124)I) for cell detection and thallium 201 (or ammonia 13) for myocardial delineation. In a subset of animals, high resolution ex vivo SPECT scans of explanted hearts were obtained to confirm that in vivo signals were derived from the cell injection site. RESULTS: NIS expression in rCDCs did not affect cell viability and proliferation. NIS activity was verified in isolated transduced cells by measuring (99m)Tc uptake. NIS(+) rCDCs were visualized in vivo as regions of (99m)Tc or (124)I uptake within a perfusion deficit in the SPECT and PET images, respectively. Cells could be visualized by SPECT up to 6 days post-injection. Ex vivo SPECT confirmed that in vivo (99m)Tc signals were localized to the cell injection sites. CONCLUSIONS: Ectopic NIS expression allows noninvasive in vivo stem cell tracking in the myocardium, using either SPECT or PET. The general approach shows significant promise in tracking the fate of transplanted cells participating in cardiac regeneration, given its ability to observe living cells using clinically applicable imaging modalities.

Lentiviral vectors bearing the cardiac promoter of the Na+-Ca2+ exchanger report cardiogenic differentiation in stem cells.

Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have acquired a cardiac phenotype. Lentiviral vectors achieved significantly higher transduction efficiencies in CDCs than any of the nine adeno-associated viral (AAV) serotypes tested. To define the most suitable vector system for reporting cardiogenic differentiation, we compared the cell specificity of five commonly-used cardiac-specific promoters in the context of lentiviral vectors. The promoter of the cardiac sodium-calcium exchanger (NCX1) conveyed the highest degree of cardiac specificity, as assessed by transducing seven cell types with each vector and measuring fluorescence intensity by flow cytometry. NCX1-GFP-positive CDC subpopulations, demonstrating prolonged expression of a variety of cardiac markers, could be isolated and expanded in vitro. Finally, we used chemical biology to validate that lentiviral vectors bearing the cardiac NCX1-promoter can serve as a highly accurate biosensor of cardiogenic small molecules in stem cells. The ability to accurately report cardiac fate and selectively enrich for cardiomyocytes and their precursors has important implications for drug discovery and the development of cell-based therapies.

Magnetic resonance imaging overestimates ferumoxide-labeled stem cell survival after transplantation in the heart.

BACKGROUND: Stem cell labeling with iron oxide (ferumoxide) particles allows labeled cells to be detected by magnetic resonance imaging (MRI) and is commonly used to track stem cell engraftment. However, the validity of MRI for distinguishing surviving ferumoxide-labeled cells from other sources of MRI signal, for example, macrophages containing ferumoxides released from nonsurviving cells, has not been thoroughly investigated. We sought to determine the relationship between the persistence of iron-dependent MRI signals and cell survival 3 weeks after injection of syngeneic or xenogeneic ferumoxides-labeled stem cells (cardiac-derived stem cells) in rats. METHODS AND RESULTS: We studied nonimmunoprivileged human and rat cardiac-derived stem cells and human mesenchymal stem cells doubly labeled with ferumoxides and beta-galactosidase and injected intramyocardially into immunocompetent Wistar-Kyoto rats. Animals were imaged at 2 days and 3 weeks after stem cell injection in a clinical 3-T MRI scanner. At 2 days, injection sites of xenogeneic and syngeneic cells (cardiac-derived stem cells and mesenchymal stem cells) were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks (50% to 90% of initial signal). Histology (at 3 weeks) revealed the presence of iron-containing macrophages at the injection site, identified by CD68 staining, but very few or no beta-galactosidase-positive stem cells in the animals transplanted with syngeneic or xenogeneic cells, respectively. CONCLUSIONS: The persistence of significant iron-dependent MRI signal derived from ferumoxide-containing macrophages despite few or no viable stem cells 3 weeks after transplantation indicates that MRI of ferumoxide-labeled cells does not reliably report long-term stem cell engraftment in the heart.

Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens.

BACKGROUND: Ex vivo expansion of resident cardiac stem cells, followed by delivery to the heart, may favor regeneration and functional improvement. METHODS AND RESULTS: Percutaneous endomyocardial biopsy specimens grown in primary culture developed multicellular clusters known as cardiospheres, which were plated to yield cardiosphere-derived cells (CDCs). CDCs from human biopsy specimens and from comparable porcine samples were examined in vitro for biophysical and cytochemical evidence of cardiogenic differentiation. In addition, human CDCs were injected into the border zone of acute myocardial infarcts in immunodeficient mice. Biopsy specimens from 69 of 70 patients yielded cardiosphere-forming cells. Cardiospheres and CDCs expressed antigenic characteristics of stem cells at each stage of processing, as well as proteins vital for cardiac contractile and electrical function. Human and porcine CDCs cocultured with neonatal rat ventricular myocytes exhibited biophysical signatures characteristic of myocytes, including calcium transients synchronous with those of neighboring myocytes. Human CDCs injected into the border zone of myocardial infarcts engrafted and migrated into the infarct zone. After 20 days, the percentage of viable myocardium within the infarct zone was greater in the CDC-treated group than in the fibroblast-treated control group; likewise, left ventricular ejection fraction was higher in the CDC-treated group. CONCLUSIONS: A method is presented for the isolation of adult human stem cells from endomyocardial biopsy specimens. CDCs are cardiogenic in vitro; they promote cardiac regeneration and improve heart function in a mouse infarct model, which provides motivation for further development for therapeutic applications in patients.

Chronic treatment with allopurinol boosts survival and cardiac contractility in murine postischemic cardiomyopathy.

Oxidative stress is a hallmark of systemic illnesses, including heart failure. Nevertheless, the overall importance of radical production in the heart remains conjectural; is it merely a marker of illness, or can intervention alter the progression of disease? This question was addressed by blocking xanthine oxidase (XO), a superoxide-generating enzyme that is upregulated in animal models of heart failure. In a randomized prospective trial design, we administered the XO inhibitor allopurinol orally to mice that had undergone massive myocardial infarction (MI). Cardiac XO activity was elevated in untreated mice after MI; allopurinol suppressed the XO activity to levels comparable to those in sham-operated mice. Eighty-one percent of untreated mice died of advanced heart failure over 2 to 4 weeks of follow-up. Survival doubled in the allopurinol-treated mice, whereas cardiac contractile function (both in vivo and in isolated muscle) was markedly improved. Response to isoproterenol was restored to near-normal levels in the allopurinol group but was attenuated in untreated mice. Oxidative modifications to proteins were prevented in the allopurinol-treated mice. Our findings indicate that targeted blockade of just one source of oxidants, XO, impacts dramatically on the progression of postischemic cardiomyopathy in mice and prevents oxidative protein modifications.

Selective contractile dysfunction of left, not right, ventricular myocardium in the SHHF rat.

The progression of hypertension to cardiac failure involves systemic changes that may ultimately affect contractility throughout the heart. Spontaneous hypertensive heart failure (SHHF) rats have depressed left ventricular (LV) function, but right ventricular (RV) dysfunction is less well characterized. Ultrathin (87 +/- 5 mircom) trabeculae were isolated from end-stage failing SHHF rats and from age-matched controls. Under near-physiological conditions (1 mM Ca(2+), 37 degrees C, 4 Hz), developed force (in mN/mm(2)) was not significantly different in SHHF LV and RV trabeculae and those of controls. SHHF LV preparations displayed a negative force-frequency behavior (40 +/- 7 vs. 23 +/- 4 mN/mm(2), 2 vs. 7 Hz); this relationship was positive in SHHF RV preparations (27 +/- 5 vs. 40 +/- 6 mN/mm(2)) and controls (32 +/- 6 vs. 44 +/- 9 mN/mm(2)). The response to isoproterenol (10(-6) M, 4 Hz) was depressed in SHHF LV preparations. The inotropic response to hypothermia was lost in SHHF LV trabeculae but preserved in SHHF RV trabeculae. Intracellular calcium measurements revealed impaired calcium handling at higher frequencies in LV preparations. We conclude that in end-stage failing SHHF rats, RV function is only marginally affected, whereas a severe contractile dysfunction of LV myocardium is present.

Physiological determinants of contractile force generation and calcium handling in mouse myocardium.

Despite the fact that the mouse has become a common tool to study cardiac dysfunction, little is known regarding the regulation of murine cardiac contractility. We have investigated the three main mechanisms that regulate cardiac output (frequency-dependent activation, length-dependent activation, and beta-adrenergic stimulation) in ultra-thin right ventricular (RV) trabeculae from the mouse heart at body temperature (37 degrees C). [Ca(2+)](i) was recorded in a subset of trabeculae iontophoretically loaded with fura-2, and rapid cooling contractures were performed to estimate the sarcoplasmic reticulum (SR) calcium load. The force-frequency relationship was positive (2-12Hz); force increased, albeit slightly, while relaxation timing decreased. As expected, in response to beta-adrenergic stimulation, force development increased while contractile duration decreased, and increased muscle length led to increased force generation. Changes in SR calcium load and the calcium transient amplitude paralleled effects on active force generation. Despite several qualitative similarities with other mammalian species, the reserve for augmentation of force via either increased frequency or beta-adrenergic stimulation was considerably smaller in mouse than in other animals. Therefore, changes in preload, as opposed to increased HR or adrenergic tone, appears to be a much more important determinant of cardiac performance in the mouse than in larger mammals.