We have developed a mathematical model of the mouse ventricular myocyte action potential (AP) from voltage-clamp data of the underlying currents and Ca2+ transients. Wherever possible, we used Markov models to represent the molecular structure and function of ion channels. The model includes detailed intracellular Ca2+ dynamics, with simulations of localized events such as sarcoplasmic Ca2+ release into a small intracellular volume bounded by the sarcolemma and sarcoplasmic reticulum. Transporter-mediated Ca2+ fluxes from the bulk cytosol are closely matched to the experimentally reported values and predict stimulation rate-dependent changes in Ca2+ transients. Our model reproduces the properties of cardiac myocytes from two different regions of the heart: the apex and the septum. The septum has a relatively prolonged AP, which reflects a relatively small contribution from the rapid transient outward K+ current in the septum. The attribution of putative molecular bases for several of the component currents enables our mouse model to be used to simulate the behavior of genetically modified transgenic mice.

It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which are thought to be due to alterations in the extracellular matrix. We tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging, which could contribute to the global changes in LV diastolic dysfunction. We used atomic force microscopy (AFM), which determines cellular mechanical property changes at nanoscale resolution in myocytes, from young (4 mo) and old (30 mo) male Fischer 344 x Brown Norway F1 hybrid rats. A measure of stiffness, i.e., apparent elastic modulus, was determined by analyzing the relationship between AFM indentation force and depth with the classical infinitesimal strain theory and by modeling the AFM probe as a blunted conical indenter. This is the first study to demonstrate a significant increase (P < 0.01) in the apparent elastic modulus of single, aging cardiac myocytes (from 35.1 +/- 0.7, n = 53, to 42.5 +/- 1.0 kPa, n = 58), supporting the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts resides, in part, at the level of the myocyte.

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Abstract-To test the hypothesis that persistent myocardial stunning can lead to hibernating myocardium, 13 pigs were chronically instrumented, and persistent stunning was induced regionally by 6 repetitive episodes of 90-minute coronary stenosis (CS) (30% reduction in baseline coronary blood flow [CBF]) followed by full reperfusion every 12 hours. During the 1st CS, CBF fell from 43Ϯ2 to 31Ϯ2 mL/min, and anterior wall thickening (AWT) fell by 54Ϯ8%, but posterior WT did not change. AWT never recovered fully and remained depressed by 31Ϯ7% before the 6th CS, reflecting persistent myocardial stunning, but baseline CBF was not changed. Surprisingly, during the 6th CS, AWT did not fall further despite a similar reduction in CBF during CS, as occurred with the 1st episode. Regional MV O 2 fell similarly during the 1st and 6th CS. During the 1st CS, plasma glucose uptake increased, whereas free fatty acid (FFA) uptake was reduced. Before the 6th CS, glucose uptake remained elevated, whereas FFA uptake remained reduced. Histology revealed enhanced glycogen deposition, which could be explained by decreased glycogen synthase kinase (GSK)-3␤ protein levels and activity. These results indicate that persistent stunning, even in the absence of chronic ischemia, can recapitulate the phenotype of myocardial hibernation. This results in a shift in the flow/function relationship where a 30% decrease in CBF is no longer accompanied by a fall in myocardial function, which could be explained, in part, by a shift in substrate utilization. These hemodynamic/metabolic adjustments could facilitate survival of hibernating myocardium. Key Words: hibernating myocardium Ⅲ myocardial stunning Ⅲ metabolism Ⅲ glycogen synthase kinase-3␤ Ⅲ ischemia M yocardial stunning is defined as the impaired but reversible reduction of contractile function after a brief ischemic episode, where the flow/function relationship is altered. 1,2 The related concept of myocardial hibernation is based primarily on clinical observations 3-6 and has been thought to involve a self-protective downregulation in myocardial function and metabolism to match the reduced O 2 supply, rather than a change in the flow/function relationship. Studies in patients with hibernating myocardium where blood flow was measured with positron emission tomography (PET) 7-9 and a study in conscious pigs with progressive coronary stenosis induced by an ameroid constrictor for one month 10 all found maintained myocardial blood flow in the face of chronically and severely reduced regional myocardial function, reminiscent of myocardial stunning. The results from these studies raised the possibility that persistent stunning might be a mechanism involved in mediating hibernating myocardium, alternative to the mechanism of downregulated myocardial blood flow and O 2 consumption. If this is found to be true, then alternative protective mechanisms must be sought to understand how hibernating myocardium can survive in the face of persistent ischemia.In order to examine whether persistent myocardial s...

Abstract-The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca 2ϩ responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca 2ϩ handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, PϽ0.01, in stunned myocytes (6.3Ϯ0.4%) compared with normal myocytes (8.8Ϯ0.4%). The time for 70% relaxation was prolonged, PϽ0.01, in stunned myocytes (131Ϯ8 ms) compared with normal myocytes (105Ϯ5 ms). The impaired contractile function was associated with decreased Ca 2ϩ transients (stunned, 0.33Ϯ0.04 versus normal, 0.49Ϯ0.05, PϽ0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca 2ϩ -current density (stunned, Ϫ4.8Ϯ0.4 versus normal, Ϫ6.6Ϯ0.4 pA/pF, PϽ0.01). There were no differences in TnI, sarcoplasmic reticulum Ca 2ϩ ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca 2ϩ transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca 2ϩ handling and dephosphorylation of phospholamban, but not TnI degradation.

Left Ventricular (LV) myocytes were isolated from 15-wk-old male mice bearing the Arg403 → Gln α-cardiac myosin heavy chain missense mutation (α-MHC403/+), a model of familial hypertrophic cardiomyopathy. LV myocytes were classified morphologically: type I, rod shaped with parallel myofibrils; type II, irregularly shaped, shorter and wider than wild-type (WT) control cells, with parallel myofibrils; and type III, irregularly shaped with disoriented myofibrils. Compared with WT myocytes, α-MHC403/+ myocytes had fewer type I cells (WT = 74 ± 3%, α-MHC403/+ = 41 ± 4%, P < 0.01) and more type III cells (WT= 12 ± 3%, α-MHC403/+ = 49 ± 7%, P < 0.01). In situ histology also demonstrated marked myofibrillar disarray in the α-MHC403/+ hearts. With the use of video edge detection, myocytes were paced at 1 Hz (37°C) to determine the effects of the mutation on myocyte function. End-diastolic length was reduced in mutant myocytes, but fractional shortening (% contraction) and sarcomere length were not. Velocity of contraction (−d L/d t max) was depressed in mutant cells, but more in type II and III cells (−31%) than in type I cells (−18%). Velocity of relaxation (+d L/d t) was also depressed more in type II and III cells (−38%) than in type I cells (−16%). Using fura 2 dye with intracellular Ca2+ transients, we demonstrated that in α-MHC403/+ myocytes, the amplitude of the Ca2+ signal during contraction was unchanged but that the time required for decay of the signal to decrease 70% from its maximum was delayed significantly (WT = 159 ± 8 ms; α-MHC403/+ = 217 ± 14 ms, P < 0.01). Sarco(endo)plasmic reticulum Ca2+-ATPase mRNA levels in α-MHC403/+ and WT mice were similar. These data indicate that the altered cardiac dysfunction of α-MHC403/+ myocytes is directly due to defective myocyte function rather than to secondary changes in global cardiac function and/or loading conditions.

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These results show that the cardiomyopathy developed by G(s)alpha x403 mice is synergistic rather than additive, most likely owing to the elevated baseline function combined with enhanced responsiveness to sympathetic stimulation.