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We hypothesized that a functional apolipoprotein LI (APOL1)-miR193a axis (inverse relationship) preserves, but disruption alters, the podocyte molecular phenotype through the modulation of autophagy flux. Podocyte-expressing APOL1G0 (G0-podocytes) showed downregulation but podocyte-expressing APOL1G1 (G1-podocytes) and APOL1G2 (G2-podocytes) displayed enhanced miR193a expression. G0-, G1-, and G2-podocytes showed enhanced expression of light chain (LC) 3-II and beclin-1, but a disparate expression of p62 (low in wild-type but high in risk alleles). G0-podocytes showed enhanced, whereas G1- and G2-podocytes displayed decreased, phosphorylation of Unc-51-like autophagy-activating kinase (ULK)1 and class III phosphatidylinositol 3-kinase (PI3KC3). Podocytes overexpressing miR193a (miR193a-podocytes), G1, and G2 showed decreased transcription of PIK3R3 (PI3KC3′s regulatory unit). Since 3-methyladenine (3-MA) enhanced miR193a expression but inhibited PIK3R3 transcription, it appears that 3-MA inhibits autophagy and induces podocyte dedifferentiation via miR193a generation. miR193a-, G1-, and G2-podocytes also showed decreased phosphorylation of mammalian target of rapamycin (mTOR) that could repress lysosome reformation. G1- and G2-podocytes showed enhanced expression of run domain beclin-1-interacting and cysteine-rich domain-containing protein (Rubicon); however, its silencing prevented their dedifferentiation. Docking, protein-protein interaction, and immunoprecipitation studies with antiautophagy-related gene (ATG)14L, anti-UV radiation resistance‐associated gene (UVRAG), or Rubicon antibodies suggested the formation of ATG14L complex I and UVRAG complex II in G0-podocytes and the formation of Rubicon complex III in G1- and G2-podocytes. These findings suggest that the APOL1 risk alleles favor podocyte dedifferentiation through blockade of multiple autophagy pathways.

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms’ tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.

ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT1R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT1R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT1R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT1R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT1R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT1R-BLK. We conclude that AT1R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)2D-deficient states; on the other hand, AT1R-BLK contributes to liganded VDR expression in 1,25(OH)2D-sufficient states.

Oxidative stress has been implicated to contribute to HIV-induced kidney cell injury; however, the role of p53, a modulator of oxidative stress, has not been evaluated in the development of HIV-associated nephropathy (HIVAN). We hypothesized that mammalian target of rapamycin (mTOR) may be critical for the induction of p53-mediated oxidative kidney cell injury in HIVAN. To test our hypothesis, we evaluated the effect of an mTOR inhibitor, rapamycin, on kidney cell p53 expression, downstream signaling, and kidney cell injury in both in vivo and in vitro studies. Inhibition of the mTOR pathway resulted in downregulation of renal tissue p53 expression, associated downstream signaling, and decreased number of sclerosed glomeruli, tubular microcysts, and apoptosed and 8-hydroxy deoxyguanosine (8-OHdG)-positive (+ve) cells in Tg26 mice. mTOR inhibition not only attenuated kidney cell expression of p66ShcA and phospho-p66ShcA but also reactivated the redox-sensitive stress response program in the form of enhanced expression of manganese superoxide dismutase (MnSOD) and catalase. In in vitro studies, the mTOR inhibitor also provided protection against HIV-induced podocyte apoptosis. Moreover, mTOR inhibition downregulated HIV-induced podocyte (HP/HIV) p53 expression. Since HP/HIV silenced for mTOR displayed a lack of expression of p53 as well as attenuated podocyte apoptosis, this suggests that mTOR is critical for kidney cell p53 activation and associated oxidative kidney cell injury in the HIV milieu.

Opiates have been reported to induce T cell loss. We evaluated the role of vitamin D receptor (VDR) and the activation of the renin-angiotensin system (RAS) in morphine-induced T cell loss. Morphine-treated human T cells displayed downregulation of VDR and the activation of the RAS. On the other hand, a VDR agonist (EB1089) enhanced T cell VDR expression both under basal and morphine-stimulated states. Since T cells with silenced VDR displayed the activation of the RAS, whereas activation of the VDR was associated with downregulation of the RAS, it appears that morphine-induced T cell RAS activation was dependent on the VDR status. Morphine enhanced reactive oxygen species (ROS) generation in a dose-dependent manner. Naltrexone (an opiate receptor antagonist) inhibited morphine-induced ROS generation and thus, suggested the role of opiate receptors in T cell ROS generation. The activation of VDR as well as blockade of ANG II (by losartan, an AT1 receptor blocker) also inhibited morphine-induced T cell ROS generation. Morphine not only induced double-strand breaks (DSBs) in T cells but also attenuated DNA repair response, whereas activation of VDR not only inhibited morphine-induced DSBs but also enhanced DNA repair. Morphine promoted T cell apoptosis; however, this effect of morphine was inhibited by blockade of opiate receptors, activation of the VDR, and blockade of the RAS. These findings indicate that morphine-induced T cell apoptosis is mediated through ROS generation in response to morphine-induced downregulation of VDR and associated activation of the RAS.

Reactive oxygen species (ROS) have been demonstrated to contribute to HIV-induced tubular cell injury. We hypothesized that HIV-induced ROS generation may be causing tubular cell injury through downregulation of vitamin D receptor (VDR) and associated downstream effects. In the present study, HIV not only downregulated tubular cell VDR expression but also inflicted DNA injury. On the other hand, EB-1089, a VDR agonist (VD), inhibited both downregulation of VDR and tubular cell DNA injury in the HIV milieu. H2O2(an O−donor) directly downregulated tubular cell VDR, whereas catalase, a free radical scavenger, inhibited HIV-induced downregulation of tubular cell VDR expression. HIV also stimulated the tubular cell renin-angiotensin system (RAS) through downregulation of VDR. Because losartan (an ANG II blolcker) partially inhibited HIV-induced tubular cell ROS generation while ANG II directly stimulated tubular cell ROS generation, it appears that HIV-induced ROS production was partly contributed by the RAS activation. VD not only inhibited HIV-induced RAS activation but also attenuated tubular cell ROS generation. Tubular cells displayed double jeopardy in the HIV milieu induction of double-strand breaks and attenuated DNA repair; additionally, in the HIV milieu, tubular cells exhibited enhanced expression of phospho-p53 and associated downstream signaling. A VDR agonist and an ANG II blocker not only preserved expression of tubular cell DNA repair proteins but also inhibited induction of double-strand breaks. In in vivo studies, renal cortical sections of Tg26 mice displayed attenuated expression of VDR both in podocytes and tubular cells. In addition, renal cortical sections of Tg26 mice displayed enhanced oxidative stress-induced kidney cell DNA damage. These findings indicated that HIV-induced tubular cell downregulation of VDR contributed to the RAS activation and associated tubular cell DNA damage. However, both VD and RAS blockade provided protection against these effects of HIV.

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome- c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome- c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.

Diabetes mellitus is complicated by the development of a primary cardiomyopathy, which contributes to the excess morbidity and mortality of this disorder. The protein kinase C (PKC) family of isozymes plays a key role in the cardiac phenotype expressed during postnatal development and in response to pathological stimuli. Hyperglycemia is an activating signal for cardiac PKC isozymes that modulate a myriad of cell events including cell death and survival. The ε-isozyme of the PKC family transmits a powerful survival signal in cardiac muscle cells. Accordingly, to test the hypothesis that endogenous activation of cardiac PKC-ε will protect against hyperglycemic cell injury and left ventricular dysfunction, diabetes mellitus was induced using streptozotocin in genetically engineered mice with cardiac-specific expression of the PKC-ε translocation activator [ψε-receptors for activated C kinase (ψε-RACK)]. The results demonstrate a striking PKC-ε cardioprotective phenotype in diabetic ψε-RACK (ε-agonist) mice that is characterized by inhibition of the hyperglycemia apoptosis signal, attenuation of hyperglycemia-mediated oxidative stress, and preservation of parameters of left ventricular pump function. Hearts of diabetic ε-agonist mice exhibited selective trafficking of PKC-ε to membrane and mitochondrial compartments, phosphorylation/inactivation of the mitochondrial Bad protein, and inhibition of cytochrome c release. We conclude that activation of endogenous PKC-ε in hearts of diabetic ε-agonist mice promotes the survival phenotype, attenuates markers of oxidative stress, and inhibits the negative inotropic properties of chronic hyperglycemia.

Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent.

In an attempt to elucidate the effects of two major risk factors of heart failure in humans, high blood pressure and coronary artery disease, renal hypertension and coronary artery constriction were induced singularly and in combination in rats, and the functional, structural, and biochemical alterations of the myocardium were examined 12-13 wk later. Renal hypertension (RH), coronary narrowing (CN), and their association (NH) resulted in left ventricular failure demonstrated by a significant increase in left ventricular end-diastolic pressure, a decrease in +dP/dt and -dP/dt, and a reduction in stroke volume and cardiac output. Measurements of ventricular loading documented that RH was characterized by elevations in systolic and diastolic wall stress of 42 and 160%, respectively. Corresponding changes with NH were 80 and 315%. CN was accompanied by an augmentation of diastolic wall stress only (280%). The abnormalities in mural stress were coupled with reductions in systolic and diastolic wall thickness-to-chamber radius ratios of 39 and 29% after CN. These anatomic parameters were preserved with RH, whereas the systolic wall thickness-to-chamber radius ratio was reduced 31% with NH. Structurally, multiple foci of replacement fibrosis were found with each intervention. The sites of tissue injury and their volume percent in the myocardium were comparable with CN and RH but were significantly more numerous and occupied a larger fraction of the ventricular wall in the presence of NH. Biochemically, the calcium dose-response curve of myofibrillar Mg2+ adenosinetriphosphatase (ATPase) activity did not vary with CN, RH, and NH. In contrast, a marked decrease in Ca2+ myosin ATPase activity was found in NH rats in association with a shift in myosin isoenzymes from V1 to V3. In conclusion, multiple physiological, morphological, and biochemical factors may participate in the generation of the abnormalities in ventricular loading with hypertension and/or coronary artery stenosis.

To determine the effects of chronic nonocclusive coronary constriction on cardiac hemodynamics, myocardial structure, and contractile protein enzyme activity, the left coronary artery was narrowed in rats, and measurements of ventricular pump function, extent and localization of tissue damage, and myofibrillar Mg2+ and Ca2+ myosin adenosinetriphosphatase (ATPase) activities were measured 3 mo later. In the presence of coronary artery stenosis averaging 56%, two different degrees of depression in global cardiac performance were identified, and the animals were divided in two groups. In the first group, left ventricular end-diastolic pressure (LVEDP) was increased and LV+ and/or--the first derivative of LV pressure (dP/dt) were decreased, whereas in the second group end-diastolic and peak systolic LV pressures, LV+ and -dP/dt and right ventricular dynamics were all impaired. Thus left ventricular dysfunction and failure occurred with coronary narrowing. Structurally, multiple foci of replacement fibrosis were found across the left ventricular wall, but the number of these lesion profiles was 2.6-fold larger in failing animals than in rats with cardiac dysfunction. Biochemically, Mg(2+)-ATPase activity in myofibrils and Ca2+ myosin ATPase were not altered biventricularly. On the other hand, a shift from V1 to V3 myosin isoenzymic content occurred in the failing left ventricle. In conclusion, the late impairment in ventricular pump function associated with prolonged coronary artery stenosis appears to be sustained more by the magnitude of myocardial damage than by defects in contractile protein enzyme activity.

Rats were trained with two running protocols previously demonstrated to result in enhanced cardiac performance. Control groups included free-eating sedentary animals and food-restricted animals in which the body weights were the same as the runners. Calcium binding by isolated sarcoplasmic reticulum (SR) was slightly but significantly increased in SR from runners at low but not high calcium concentrations at 15 s and 1 min. Calcium uptake in the presence of 1 mM oxalate was increased in SR from runners. Actomyosin ATPase activity was increased by 10% (P less than 0.001) with one running protocol but not with the other. Myosin Ca2+ ATPase activity and actin-activated ATPase activity were also slightly increased in hearts of runners. In food-restricted cardiac actomyosin ATPase was significantly decreased. Actomyosin ATPase activity was found to be normal in hearts of sedentary animals subjected to water immersion without exercise. Therefore, physical training of rats by running, which produces a cardiac mechanical advantage similar to training by swimming, is not accompanied by cardiac biochemical changes of the same magnitude as in the hearts of swimmers.

In rats, chronic diabetes is associated with depressed cardiac myosin ATPase activity and a shift from the predominant V1 isoenzyme to V3, correlating with depressed contractility. Rabbit myocardium consists mostly of the V3 isoenzyme, and therefore a switch to even more V3 isoenzyme in diabetes might not be possible and therefore not explain the mechanical abnormalities observed. To explore this, rabbits were made diabetic with 140-150 mg/kg of alloxan, and their hearts were studied 3 days, 1 mo, 3 mo, and 6 mo later. Ca2+-myosin-ATPase activity was decreased in the diabetic rabbit at 1, 3, and 6 mo, correlating with increased percent V3. Actin-activated Mg2+-ATPase activity was not significantly decreased in diabetics, but myofibrillar ATPase activity was decreased in 6-mo diabetic animals. When 3- to 4-mo diabetic animals were administered insulin for 3-4 additional months, myosin-ATPase activity and isoenzyme distribution normalized. These results correlate well with mechanical changes in papillary muscle from these same hearts. They suggest that in rabbit, as in rat, changes in cardiac contractile function are at least partially mediated by changes in myosin isoenzyme composition and are reversible with insulin.

The purpose of this study was to determine whether a chronic swimming program could reverse the decreased cardiac function and altered myosin biochemistry found in hearts of rats with established renal hypertension. Ten wk after the onset of hypertension [midpoint (m)], hearts from normotensive controls (C) and hypertensives (H) were studied in an isolated working heart apparatus, and myosin biochemistry was analyzed. Half of the control and hypertensive animals were then subjected to a 10-wk swimming program (Sw) and their hearts were compared with those from age-matched sedentary rats. Body weight was no different at the midpoint of the study between Cm and Hm or at the end point (e) of the study among Ce, Swe, He, or H-Swe. Swimming had no effect on blood pressure in either normotensive or hypertensive rats. Dry heart weight was increased by 46% in Hm compared with Cm and by 36% in He, 21% in Swe, and 61% in H-Swe when compared with Ce. Hypertension was associated in both the mid- and end-point studies, with decreases in coronary flow, stroke work (both per gram left ventricle), ejection fraction, and midwall fractional shortening. In addition, actin-activated myosin adenosinetriphosphatase (ATPase) activity was decreased in Hm and He associated with an increase in the content of the V3 myosin isoenzyme. Although the coronary deficit was not corrected in H-Swe, stroke work, ejection fraction, and fractional midwall shortening were normalized compared with control hearts. Myosin ATPase activity and the myosin isoenzyme distribution were similarly restored in H-Swe.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the effects of graded insulin therapy on cardiac function and biochemistry, rats were made diabetic by streptozotocin (50 mg/kg) and subsequently treated with either 3 U of insulin per day (D3) or 5 U/day (D5) and compared with untreated diabetic rats (D phi) and a nondiabetic control group (C). Blood glucose, water consumption, and heart and body weights in D3 and D5 showed dose-dependent responses between those of D phi and C. Cardiac function was studied at similar heart rates and similar left atrial and aortic pressures in an isolated working heart apparatus. Hearts from D phi showed significant decreases in end-diastolic pressure, peak left ventricular systolic pressure, and positive dP/dt, whereas these values in D3 and D5 were similar to those in C. The isovolumic relaxation period was significantly longer in the D phi group, intermediate between D phi and C in D3, and the same in D5 and C. Ca2+-ATPase activity of myosin and actin-activated Mg2+-ATPase activity was depressed in D phi, partially corrected in D3, and completely corrected in D5. Myosin isoenzyme distribution displayed a shift from the predominant V1 pattern observed in C to a predominant V3 pattern in D phi. Treatment with 3 U of insulin per day partially corrected the isoenzyme abnormality, and treatment with 5 U/day restored the isoenzyme distribution to normal. These results indicate that gross cardiac contractile function can be normalized with insulin dosages that are not sufficient to correct hyperglycemia, polydipsia, or body and heart weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The intrinsic myocardial adaptation to chronic beta-blockade was explored in normal male Wistar rats. Propranolol was administered by subcutaneous infusion using osmotic minipumps for 3–4 wk (P group). Heart rate fell by approximately 100 beats/min. A second group of animals was similarly treated but had their pumps removed several days before study (P-R group). Heart rate rose, but remained below base line. Study of isolated ventricular papillary muscle from P, P-R, and age-matched controls (C group) revealed prolonged contraction duration in P and P-R groups, but no change in shortening velocity. A greater shortening of time to peak tension and time to one-half relaxation in response to norepinephrine was demonstrated in P and P-R groups. Acute in vivo or in vitro administration of propranolol had no effect on base-line mechanical performance. No changes in the Ca2+, actin-activated Mg2+ myosin adenosine triphosphatase (ATPase) activity, or myosin isoenzyme distribution were found. Free thyroxine levels were decreased in P but not P-R groups. These findings indicate that chronic propranolol therapy in normal rats slows contraction and relaxation without altering contractility. The greater shortening of contraction duration in response to norepinephrine is partly offset by the prolongation of the base-line contraction.

To study effects of physiologic hypertrophy on contractile protein ATPases and sarcoplasmic reticulum, hypertrophy was caused in female Wistar rats by a chronic swimming program. Nonhypertrophied hearts of female control sedentary rats and rats made to run on a treadmill program were also examined. The swimming program, but not the running program, resulted in a significant increase in heart weight. Actomyosin ATPase activity was also increased by 15% in the hearts of swimmers but not runners. Similar increases were observed for Ca2+-activated myosin ATPase activity and actin-activated ATPase of myosin. Sarcoplasmic reticulum from the hearts of swimmers showed increased calcium binding and calcium uptake as a function of time and of calcium concentration. Sarcoplasmic reticulum ATPase activities were not altered by hypertrophy. These findings in physiologic hypertrophy contrast with those of pathologic hypertrophy in which ATPase activity of contractile proteins and calcium binding and uptake of sarcoplasmic reticulum have generally been found to be depressed.

We have previously shown that swim conditioning corrects the depressed mechanical function and myosin adenosinetriphosphatase (ATPase) activities associated with renovascular hypertension (HTN) in the rat. The present study was designed to assess the effects of swim conditioning on another form of systolic overload, subdiaphragmatic suprarenal aortic stenosis. Cardiac mechanics in an isolated working heart apparatus and myosin enzymology were studied in four groups of rats: controls (C), animals with chronic systolic overload secondary to aortic constriction (St), swim-conditioning animals (Sw), and animals exposed to a combined load (St-Sw). Heart weight was increased by 23% in St, 27% in Sw, and 36% in St-Sw. In contrast to HTN, cardiac pump and muscle function were not depressed in St. Sw was associated with improved cardiac output, stroke work, and velocity of circumferential fiber shortening. St-Sw showed improved mechanical cardiac performance relative to both C and St. The percent of ventricular myosin of the V1 type and Ca2+-activated myosin ATPase activity relative to C was unchanged in Sw but was depressed in St and St-Sw. These data demonstrate that the salutory mechanical effects of Sw can be superimposed on the systolic overload of St. However, the dissociation between mechanics and myosin enzymology suggests that factors in excitation-contraction coupling other than myosin isoenzyme shifts are responsible for this finding.