Attenuated sarcomere lengthening of the aged murine left ventricle observed using two-photon fluorescence microscopy.

The Frank-Starling mechanism, whereby increased diastolic filling leads to increased cardiac output, depends on increasing the sarcomere length (Ls) of cardiomyocytes. Ventricular stiffness increases with advancing age, yet it remains unclear how such changes in compliance impact sarcomere dynamics in the intact heart. We developed an isolated murine heart preparation to monitor Ls as a function of left ventricular pressure and tested the hypothesis that sarcomere lengthening in response to ventricular filling is impaired with advanced age. Mouse hearts isolated from young (3-6 mo) and aged (24-28 mo) C57BL/6 mice were perfused via the aorta under Ca(2+)-free conditions with the left ventricle cannulated to control filling pressure. Two-photon imaging of 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium fluorescence was used to monitor t-tubule striations and obtain passive Ls between pressures of 0 and 40 mmHg. Ls values (in µm, aged vs. young, respectively) were 2.02 ± 0.04 versus 2.01 ± 0.02 at 0 mmHg, 2.13 ± 0.04 versus 2.23 ± 0.02 at 5 mmHg, 2.21 ± 0.03 versus 2.27 ± 0.03 at 10 mmHg, and 2.28 ± 0.02 versus 2.36 ± 0.01 at 40 mmHg, indicative of impaired sarcomere lengthening in aged hearts. Atomic force microscopy nanoindentation revealed that intact cardiomyocytes enzymatically isolated from aged hearts had increased stiffness compared with those of young hearts (elastic modulus: aged, 41.9 ± 5.8 kPa vs. young, 18.6 ± 3.3 kPa; P = 0.006). Impaired sarcomere lengthening during left ventricular filling may contribute to cardiac dysfunction with advancing age by attenuating the Frank-Starling mechanism and reducing stroke volume.

Dantrolene suppresses spontaneous Ca2+ release without altering excitation-contraction coupling in cardiomyocytes of aged mice.

Cardiac dysfunction in the aged heart reflects abnormalities in cardiomyocyte Ca(2+) homeostasis including altered Ca(2+) cycling through the sarcoplasmic reticulum (SR). The ryanodine receptor antagonist dantrolene exerts antiarrhythmic effects by preventing spontaneous diastolic Ca(2+) release from the SR. We tested the hypothesis that dantrolene prevents spontaneous Ca(2+) release without altering excitation-contraction coupling in aged myocardium. Left ventricular cardiomyocytes isolated from young (3 to 4 mo) and aged (24-26 mo) C57BL/6 mice were loaded with the Ca(2+) indicator fluo-4. Amplitudes of action potential-induced Ca(2+) transients at 1-Hz pacing were similar between young and aged mice, yet cell shortening was impaired in aged mice. Isoproterenol (1 µM) increased Ca(2+) transient amplitude and cell shortening to identical levels in young and aged; dantrolene (1 µM) had no effect on Ca(2+) transients or cell shortening during pacing. Under Ca(2+) overload conditions induced with 10 mM extracellular Ca(2+) concentration, spontaneous Ca(2+) waves were of diminished amplitude and associated with lower SR Ca(2+) content in aged versus young mice. Despite no effect in young mice, dantrolene increased SR Ca(2+) content and Ca(2+) wave amplitude in aged mice. In the presence of isoproterenol following rest from 1-Hz pacing, Ca(2+) spark frequency was elevated in aged mice, yet the time to spontaneous Ca(2+) wave was similar between young and aged mice; dantrolene decreased Ca(2+) spark frequency and prolonged the time to Ca(2+) wave onset in aged mice with no effect in young mice. Thus dantrolene attenuates diastolic Ca(2+) release in the aged murine heart that may prove useful in preventing cardiac dysfunction.

Genetic manipulation of the cardiac mitochondrial phosphate carrier does not affect permeability transition.

The Mitochondrial Permeability Transition (MPT) pore is a voltage-sensitive unselective channel known to instigate necrotic cell death during cardiac disease. Recent models suggest that the isomerase cyclophilin D (CypD) regulates the MPT pore by binding to either the F0F1-ATP synthase lateral stalk or the mitochondrial phosphate carrier (PiC). Here we confirm that CypD, through its N-terminus, can directly bind PiC. We then generated cardiac-specific mouse strains overexpressing or with decreased levels of mitochondrial PiC to assess the functionality of such interaction. While PiC overexpression had no observable pathologic phenotype, PiC knockdown resulted in cardiac hypertrophy along with decreased ATP levels. Mitochondria isolated from the hearts of these mouse lines and their respective non-transgenic controls had no divergent phenotype in terms of oxygen consumption and Ca(2+)-induced MPT, as assessed by swelling and Ca(2+)-retention measurements. These results provide genetic evidence indicating that the mitochondrial PiC is not a critical component of the MPT pore.